Glucan drug delivery system and adjuvant

ABSTRACT

The invention describes a whole β-glucan drug delivery vehicle that non-specifically enhances the immune response, and is safe for human use. A drug is incorporated into a whole β-glucan microparticle, and the combination is administered to an individual. The β-glucan vehicle allows sustained release of the drug component while simultaneously enhancing the effectiveness of the drug by boosting the individual&#39;s endogenous immune response.

RELATED APPLICATIONS

This application claims priority to PCT application PCT/US90/03440,filed Jun. 14, 1990, which is a continuation-in-part of U.S. Ser. No.366,490, filed Jun. 15, 1989, now issued as U.S. Pat. No. 5,032,401.

BACKGROUND OF THE DISCLOSURE

Advances in biotechnology and immunology have presented new challengesfor obtaining safe and effective drugs, such as vaccines. For example,new generation subunit and antiidiotype antigens yield very safevaccines; however, these vaccines, in general, provide poor immunestimulation and prophylactic effects. Therefore, an important aspect ofany new drug or vaccine formulation is a component that enhances itssafety and efficacy by providing a delivery mechanism and, in the caseof vaccines, by boosting the immune response to the antiget. Adjuvantscan generally be categorized as components that boost the immuneresponse, and as delivery systems that enhance antigen presentation,provide sustained release of the drug or antiget for extended periods,or target the drug or antiget to specific immune cells.

Serious drawbacks exist in many of today's adjuvants and deliverysystems. Most are crude preparations of bacterial or plant origin, oroil emulsion systems, the active components and modes of action of whichare unknown. In addition, these compounds are usually toxic and cannotbe used safely, especially for human applications. Some preparations ofthe yeast cell wall component. β-glucan, have been shown to provideenhanced resistance to several infectious diseases when given inconjunction with viral vaccines or killed infected cells. Reynolds etal., 1980, Infect. Immunity, 30:51-57; Holbrook et al., 1981, Infect.Immunity, 35:534-546; Benach et al., 1982, Infect. Immunity, 36:947-951.Some of the adverse effects of administering other β-glucan preparationsare described by Williams et al. in U.S. Pat. No. 4,761,402. Theseeffects include anaphylaxis, granuloma development, hypotensiondevelopment and a high degree of acute toxicity.

SUMMARY OF THE INVENTION

The invention relates to a novel pharmaceutical composition which is adrug delivery vehicle and which nonspecifically enhances the immuneresponse. The composition comprises whole glucan particles and apharmacologically active substance, such as a drug or antigert. The drugor antigen can be contained within, uniformly dispersed with, orchemically linked to the whole glucan particles.

Methods for utilizing whole glucan particles in pharmaceuticalformulations which provide, in combination, (1) the prolonged release ofthe drug; (2) longer half-life of the drug by protecting it fromproteolytic, hydrolyric and other clearance mechanisms; (3) targeteddelivery of the drug to macrophages; and (4) stimulation of the immuneresponse are also the subject of the present invention.

When the present composition is administered to an individual theentrapped drug is released through the glucan matrix into thephysiological environment. Where the drug is an antigen, the β-glucancomponent simultaneously acts as an adjuvant to the antigen by enhancingthe immune response in the individual.

Whole glucan particles are very pure preparations of β-glucan moleculeswhich avoid many of the undesirable side effects associated with lesspure preparations. Whole glucan particles retain the in vivothree-dimensional morphology of the yeast cell walls from which they arederived. Thus, the particles are hollow, which allows the drug orantiget to be incorporated into the cavity. Whole glucan particles havea higher water-holding capacity than β-glucans prepared by other methodswhich disrupt the cell walls. The water-holding capacity can becontrolled by modifying the β-glucan structure, for example, bymodifying the amount of branching.

The invention further provides a method for providing a drug or antigento an individual while simultaneously providing an adjuvant to boost theimmune response to the drug or antigen, by administering to theindividual a drug contained within (e.g., encapsulated or entrapped), oruniformly dispersed in, or chemically linked to whole glucan particles.For example, a vaccine can be incorporated into a whole glucan particle,and the particle administered to an individual to protect against aninfectious disease, while simultaneously boosting the individual'simmune response to the vaccine.

The invention also teaches methods to incorporate drugs or antigens intothe intact whole glucan particle, thus providing substantialimprovements in the administration and efficacy of drug formulations.

This invention provides a safe, non-toxic vehicle for in vivo drugdelivery that also enhances the immune response, enhances drugpresentation in vivo and targets the drug to specific immune cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating the release rate of several proteins ofvarying molecular weight from whole glucan particles.

FIG. 2 is a graph illustrating the rate of release of bovine serumalbumin (BSA) from whole glucan particles of different permeabilities.

FIG. 3 is a bar graph illustrating the effect on release rate varyingamounts of crosslinking of the drug Cytochrome-C with whole glucanparticles.

FIG. 4 is a graph illustrating the immunological effect (antibody titer)of whole glucan particles mixed with BSA which were administered to amouse.

FIG. 5 is a graph illustrating the immunological effect (antibody Titer)of whole glucan particles mixed with BSA which were administered as abooster to a mouse.

FIG. 6 is a graph comparing the immunological effect (antibody titer) ofwhole glucan particles in which BSA is chemically cross-linked to theparticle and in which BSA is physically mixed with the particles.

FIG. 7 is a graph comparing the immunological effect (antibody titer) ofwhole glucan particles in which P55 is chemically cross-linked to theparticles and in which P55 is physically mixed with the particles.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a unique pharmaceutical composition for thecontrolled and/or continuous release of a drug or antigen from a wholeglucan particle combined with an immune system enhancement induced bythe β-glucan. The composition thus provides a drug delivery andcontrolled release system which acts as an adjuvant to the drug. Theterm "adjuvant" as used herein means a substance which is added to adrug product or formulation which prolongs and enhances the action ofthe drug or active ingredient. For example, in a vaccine formulation,the whole glucan particles provide a vehicle which enhances antigenicityto the vaccine by prolonging its half-life, targeting it to themacrophages or antigen-presenting cells and simultaneously activatingthese cells.

β-Glucans provide enhanced resistance to infectious diseases bynon-specifically activating a hosts' immune defense system. Activationoccurs through interaction with specific β-glucan receptors on monocytesthereby inducing the release of interleukin-1 (IL-1) and other cytokinesand cellular mediators. Czop, (1986) Pathology and ImmunopathologyResearch, 5:286-296; Williams et al., 1988, International Journal ofImmunopharmacology, 9:261-267.

Compositions of the present invention, comprising a whole glucanparticle and a drug or other pharmaco logically active substance, can beused to provide, in combination, a drug delivery vehicle, and anadjuvant in the administration of drugs or vaccines, which compositionsare safe and efficacious in humans and animals. The compositions are twophase systems comprising whole glucan particles and a drug or anantigen. The compositions provide substantial improvements in theadministration and efficacy of drug formulations. These improvementsinclude:

(1) Providing a non-toxic biodegradable carrier with a definedcomposition and structure;

(2) Providing a delivery vehicle capable of sustained release of thedrug or antigen component;

(3) Targeting the drug or antigen component to macrophages; and

(4) Providing a non-specific immunostimulant, with a known mode ofaction.

This combination of properties has a synergistic effect, thus, thepresent invention provides a whole glucan particle delivery system thattargets the drug or vaccine to macrophages, activates the macrophages,and extends the drug in vivo half-life by protecting it from degradation(proteolytic or hydrolyric) and rapid clearance, thus resulting inincreased potency and efficacy compared with individual formulations ofdrug, antiget or whole glucan particles.

The Terms "whole glucan", "whole glucan particles", "whole β-glucan" or"whole β-glucan particles" as used herein refer to whole β-glucanparticles. Whole β-glucan particles are essentially micron-sized hollowspheres composed of a rigid, semi-permeable glucan matrix. Wholeβ-glucan particles have the ability to swell in aqueous solutions.

Whole glucan particles are prepared from yeast cells by the extractionand purification of the alkali-insoluble glucan fraction from the yeastcell walls. The yeast cells are treated with an aqueous hydroxidesolution, without disrupting the yeast cell walls, which digests theprotein and intracellular portion of the cell, leaving the glucan wallcomponent devoid of significant protein contamination, and havingsubstantially the unaltered cell wall structure of β(1-6) and β(1-3)linked glucans. A more detailed description of whole glucan particlesand the process of preparing them is described by Jamas et al. in U.S.Pat. No. 4,810,646 and in co-pending patent applications U.S. Ser. No.166,929, U.S. Ser. No. 297,752 and U.S. Ser. No. 297,982, the teachingsof which are incorporated herein by reference.

Whole glucan particles have been shown to activate human monocytemacrophages by the same mechanisms characterized for other β-glucans.Czop, Pathology and Immunoatholog Research, 5:286-296 (1986). A uniquefeature of the whole glucan particles is that they retain the in vivo3-dimensional morphology of the yeast cell wall. Whole glucan particlesprepared by this method have several advantages over other β-glucanpreparations, such as those described by DiLuzio et al.; in theInternational Journal of Cancer, 24:773-779 and Manners et al., inBiochemistry Journal, 135:19-30 (1973): they are highly pure (e.g., haveless than one percent (w/w) protein and less than three percent (w/w)chitin and glycogen), they are intact, having a hollow spherical shapewhich allows agents to be incorporated into the particles and hey can bechemically modified (e.g., crosslinked) to regulate the release rate ofthe encapsulated drug, and the rate of degradation of the β-glucanmatrix. In addition, whole glucan particles can activate macrophagecells, thus can be used as carriers or transport vehicles foradministration of drugs or antigens to an individual, whilesimultaneously boosting the individual's immune response, therebyenhancing the action of the drug. The whole glucan carrier acts todeliver the drug or antigen directly to macrophages, where it is slowlyreleased, causing a heightened and sustained immune response. Thus, thepresent composition allows drugs or antigens to be directed or targetedto macrophage cells.

The whole glucan particles are biodegradable, that is, they bioerodeover time in a physiological environment. The terms "biodegradable" or"bioerodible" as used herein are defined as the property orcharacteristic of a body of microporous material to innocuouslydistintegrate or break down as a unit structure or entity over a periodof time, in response to the biological environment by one or morephysical or chemical degradative processes, for example by enzymaticaction, hydrolysis, dissolution. The erosion rate may be controlled byvarying the ratio of β(1-6):β(1-3) linkages in the β-glucan matrix or bycrosslinking.

The drugs suitable for use in the present composition are biologicallyactive substances. These substances include biologically activepolypepides, antigens and vaccines. Any of the drugs used to treat thebody can be incorporated in the present composition. The term "drug" isused herein in its broadest sense, as including any composition orsubstance that will produce a pharmacologic response. Suitable drugs foruse with the composition of the invention include without limitation:protein drugs such as insulin; desensitizing agents such as antigens;vaccines such as smallpox, yellow fever, distemper, cholera, fowl pox,antivenom, scarlet fever, diphtheria toxoid, tetanus toxoid, whoopingcough, influenza, rabies, mumps, measles and poliomyeliis; anibiotics,such as penicillin, tetracycline, neomycin and erythromycin;antiallergenics, steroids; decongestants; anti-cholinesterases;sedatives; tranquilizers; estrogens; humoral agents; antipsychotics;antispasmodics; antimalarials; antihistamines; cardioactive agents;nutritional agents such as vitamins, amino acids and fats. Other drugshaving the same or different physiological activity as hose recitedabove can be employed in drug delivery systems within he scope of thepresent invention. Suitable mixtures of drugs can also be incorporatedinto the composition in lieu of a single drug.

Drugs can be in various forms, such as uncharged molecules, componentsof molecular complexes, or pharmacologically acceptable salts, such ashydrochloride, hydrobromide, sulfate, phosphate, nitrate, borate,acetate, maleate, tartrate and salicylate. For acidic drugs, salts ofmetals, amines or organic cations (e.g., quaternary ammonium) can beused. Simple derivatives of the drugs (such as ethers, esters, amides),which have desirable retention and release characteristics, but whichare easily hydrolyzed by body pH or enzymes can be used.

The amount of drug incorporated in the drug delivery device varieswidely depending on the particular drug, the desired therapeutic effectand the time span for which it takes the glucan matrix to swell, erodeor dissolve. A variety of β-glucan particles are available to providecomplete dosage regimes for therapy for a range of therapeutic orprophylactic treatments, thus, there is no critical upper limit on theamount of drug incorporated into the device. The lower limit will dependupon the activity of the drug and the time span of its release from thedevice.

The present compositions are produced by causing the whole glucanparticle to swell in the presence of a solution of the drug of choice.Various drugs can therefore be incorporated into the particles bynatural diffusion. Once absorbed within the particles, these drugs maybe entrapped by removing the solvent, or by precipitation (e.g., bychange of pH, ionic environment or solvent). For example, proteinswithin the particle can be precipitated by adding ammonium sulfate,ethanol or acetone to a solution of drug and whole glucan particles. Theoutward diffusion or release rate of the entrapped drugs is therefore afunction of their rate of dissolution in the environment of use, andtheir rate of diffusion through the semipermeable glucan matrix.

Several methods can be used to swell the glucan particles. Generally, anaqueous solution of the drug to be loaded is prepared and added to anappropriate quantity of whole β-glucan particles and the mixture isallowed sufficient time (generally up to six hours) for the particles toswell. The swollen particles are then removed from the solution, anddried, or contacted with another compound to precipitate the entrappeddrug.

The release mechanism of the drug from the cavity of the whole glucanparticles into the physiological environment is through naturaldiffusion and/or degradation of the polymeric glucan network. The rateof release of the drug can be controlled by changing the ratio ofβ(1-6):β(1-3) linkages in the glucan. Methods of modifying and otherwisemanipulating the ratio of β(1-6):β(1-3) linkages, thereby altering theproperties of the β-glucan matrix, are described in detail by Jamas etal. in U.S. Pat. No. 4,810,646; and in co-pending patent applicationsU.S. Ser. No. 07/166,929; U.S. Ser. No. 07/297,752 and U.S. Ser. No.07/297,982, the teachings of which are incorporated herein by reference.For example, by chemical, enzymatic or genetic modification of the ratioof β(1-6):β(1-3) linkages, the water-holding capacity and permeabilityof the whole glucan particle can be changed, thereby controlling therate of release of the drug incorporated therein. The effect of reducingpermeability of the β-glucan matrix on the release rate of bovine serumalbumin (BSA) is illustrated in FIG. 2. Additionally, the size (e.g.,molecular weight) of the drug molecule is important. Larger molecules,such as proteins, will exhibit a slower rate of release in vivo, asillustrated in FIG. 1. Thus, the properties of the glucan carrier can betailored specifically to the drug of interest.

The release rate of a molecule from a whole glucan particle can bemodified by crosslinking it to the glucan matrix. This technique isparticularly useful for low molecular weight agents which would normallydiffuse rapidly through the glucan matrix. This can be achieved, forexample, by adding a crosslinking agent to the mixture of β-glucanparticles and the drug. The effect of crosslinking is shown in FIG. 3.

The present composition can be administered in any way commensurate withthe result or effect desired from the drug. Such methods ofadministration include orally, intramuscularly, transdermally,intradermally, intravenously, or via the gastrointestinal tract. Thecomposition can be formulated into a liquid solution, tablet, lozenge,suppository, insert or the like. One of the advantages of the presentcomposition is the degradation in vivo of the β-glucan vehicle intonon-toxic natural compounds.

The amount of he composition administered to a subject will vary on anindividual basis depending upon the drug used, the nature of thetreatment or therapy, the type and severity of the symptoms to betreated, the size and physical condition of the individual, and theresults sought.

Whole glucan particle compositions have several advantages asimmunostimulants compared to alternate materials, such as aluminumhydroxide and glucans prepared by other methods (e.g., Di Luzio et al.,Int. J. Cancer, 24:773-779 (1979); Manners et al., 1973, Biochem J.135:19-31). Whole glucan particles are more pure than these glucans andretain the in vivo, three dimensional morphology of the yeast cell,thereby providing an intact, hollow structure into which drugs can beincorporated. Glucans prepared by other methods are not intact becausethe processes used include treatments which disrupt he yeast cell walls,and which destroys the unique functional features of whole glucanparticles.

The invention is further illustrated by the following Examples.

EXAMPLE 1

Method to Incorporate Proteins Into Whole Glucan Particles by Swellingand Physical Entrapment

Three proteins of different molecular weight were incorporated intowhole glucan particles using the following procedure. Solutions ofcytochrome-C (cyt. C; Mw=14,000 daltons), ovine serum albumin (BSA;Mw=67,000 daltons) and alcohol dehydrogenase (yeast) (ADH; Mw=150,000daltons) were dissolved in deionized water at concentrations ofapproximately 12 mg/ml. One milliliter of each protein solution wasadded to 150 mg of whole glucan particles (produced from baker's yeastUniversal Foods, Wis.) and from Saccharomyces cerevisiae R4 according tothe method described by Jamas et al. in U.S. Pat. No. 4,810,646 in atest-tube and allowed to swell for two hours at room temperature. Thetubes were then transferred to a 45° C. oven and allowed to dry for 12hours. The resulting dried whole glucan particles containedapproximately 80 mg protein/gram of particles.

EXAMPLE 2

Sustained Release of Proteins from Whole Glucan Particles

Three proteins were selected to demonstrate the release rate ofdifferent sized molecules from whole glucan particles. Cytochrome-C(Mw=14,000 daltons), BSA (Mw=67,000 daltons) and ADH (Mw=150,000daltons) were loaded into whole glucan particles according to the methoddescribed in Example 1. The dried, loaded particles were resuspended in10 ml deionized water and were agitated at 37° C. Samples were removedat regular time intervals and assayed spectrophotometrically forreleased protein. FIG. 1 illustrates the diffusion kinetics of cyt-C,BSA and ADH, from the whole glucan particles, compared to the drugsalone. The amount of time to release 50% of the three drugs is shown inTable 1.

                  TABLE 1                                                         ______________________________________                                        Time to Release 50% of Protein (T.sub.50) from                                Whole Glucan Particles (Derived from Baker's                                  Yeast)                                                                                         Molecular Weight                                                                           T.sub.50                                        Protein          (daltons)    (minutes)                                       ______________________________________                                        Cytochrome-C     14,000       31                                              Bovine Serum Albumin                                                                           67,000       80                                              Alcohol Dehydrogenase                                                                          150,000      200                                             ______________________________________                                    

These results demonstrate that the release rate of the proteins from thewhole glucan particles is related to the molecular weight of theprotein.

EXAMPLE 3

Control of Release Rate From Whole Glucan Particles by Modifying thePermeability of the Glucan Membrane

The following experiment was carried out according to the methoddescribed in Example 2, except that whole glucan particles having ahigher degree of β(1-6) branching compared with other yeast strains wereused. These altered whole glucan particles were derived from a mutantstrain of yeast, Saccharomyces cerevisiae R4 (NRRL Y 15903, described inU.S. Pat. No. 4,810,646). The release rate of BSA from whole glucanparticles produced from commercial Bakers yeast and the mutant strain R4according to the procedure described in U.S. Pat. No. 4,810,646, R4 wasdetermined and compared. The results, shown in FIG. 2, demonstrate thatthe lower permeability of the glucan in particles derived from mutant R4results in longer retention times of the entrapped BSA (T₅₀ =204minutes) compared with particles derived from Bakers yeast (T₅₀ =82minutes).

EXAMPLE 4

Control of Release Rate of Small Molecules from Whole Glucan Particlesby Chemical Cross-linking

Whole glucan particles containing cross-linked cytochrome-C wereprepared by first reacting 5 mg cytochrome-C with 2.5 mg of theheterobifunctional cross-linking reagent sulfosuccinimidyl6-(4'-azido-2'-nitrophenylamino) hexanoate (sulfo-SANPAH) in 1 ml 10 mMsodium phosphate buffer pH 7.4 for 16 hours at 25° C. in the dark. Onemilliliter of the sulfoSANPAH-cytochrome-C conjugate was swelled intothe whole glucan particle cavity by mixing with 150 mg of whole glucanparticles and incubating at 25° C. for 2 hours in the dark. Thesulfo-SANPAH-cytochrome-C conjugate was cross-linked to the whole glucanparticles by exposure to bright light. The unreacted sulfo-SANPAH,cytochrome-C and sulfo-SANPAH-cytochrome-C were removed by washing thewhole glucan particles in water. The cross-linked whole glucanparticle:cytochrome-C conjugate was dried and stored at 4° C.

The release rate of the protein from the particles was determinedaccording to the method described in Example 2. FIG. 3 shows that therelease rate of cytochrome-C can be reduced to provide greater than 90%retention over a 24 hour period by increasing the amount of crosslinkeradded to the whole glucan particles containing cytochrome-C.

EXAMPLE 5

Adjuvant Effect of Whole Glucan Particles in Immunization of Mice WithBSA

The in vivo adjuvant effect of whole glucan particles in mice wasdemonstrated by an increase in antibody production in response to theantigen, BSA. BSA was incorporated into whole gucan particles asdescribed in Example 1. CD-1 mice were immunized intradermally with arange of doses of whole glucan particles mixed with BSA in phosphatebuffered saline. The dosages of whole glucan particles, containing 10 gBSA per mouse, were: 0 μg, 2 μg, 10 μg, 250 μg and 1250 μg. BSA (10 μg)alone was used as a control. Anti-BSA antibody titers were determined byELISA assay two weeks after immunization. FIG. 4 shows the antibodytiters 2 weeks post-immunization. At 3 weeks post-immunization mice wereboosted with a second injection of the same dosages, and the antibodytiters were determined at two weeks post-boost. FIG. 5 shows theantibody titers 2 weeks post-boost.

The results showed that whole glucan particles had a stimulatory effecton anti-BSA production both in the primary and secondary immuneresponses (FIGS. 4 and 5). Stimulation was observed at doses as low as 2μg whole glucan particles per animal (approximately 100 μg/kg bodyweight).

EXAMPLE 6

Combined Adjuvant and Delivery Properties of Whole Glucan Particles InMice

Based on the results of Example 5 the combined adjuvant/deliveryproperties of the whole glucan particles were investigated with aprotein antigen (BSA) and a 55-amino acid peptide (P55). BSA and P55were loaded into the hollow cavity of whole glucan particles and werecross-linked as described in Example 4. The cross-linked formulationswere prepared so that each dose (0.2 ml)consisted of 100 μg whole glucanparticles and either 10 μg BSA or 50 μg P55. Formulations containing thesame ratios of whole glucan particles and antigen were also preparedJust by mixing together the whole glucan particles with BSA or P55. Eachformulation was injected into separate groups of 5 mice (subcutaneousadministration) on day 1 of the study and the animals were boosted onday 14 with the same formulation, as described in Example 5. The animalswere sacrificed on day 26 and serum was collected and analyzed foranti-BSA or anti-P55 antibodies by direct ELISA. FIGS. 6 and 7 summarizethe results.

As observed in FIGS. 6 and 7, the utilization of the whole glucanparticles as combination adjuvants and delivery vehicle resulted insignificantly higher antibody titers than simple mixtures of the antigenwith the glucan.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

We claim:
 1. A two-phase drug composition for use to stimulate an immuneresponse in a mammal, comprising:a. a first phase comprising bioerodiblewhole β-particles which are hollow spheres comprised of a semi-permeableglucan matrix; and b. a second phase comprising an immune stimulatingdrug which is capable of being incorporated within each of the wholeglucan particles by natural diffusion;to form a two-phase drugcomposition the drug being contained within or dispersed in said wholeβ-glucan particles, and the composition being capable, when placed in aphysiological environment, of releasing the drug over time into thephysiological environment, the whole β-glucan particles beingbioerodible in response to the physiological environment which wholeβ-glucan particles stimulate an immune response in the mammal.